The progressive loss of cognitive abilities in HIV-1-infected individuals, which is currently referred to as HIV-1-associated neurocognitive disorders (HAND; also popularly known as neuroAIDS), remains a substantial clinical concern. Recent studies have shown that the disruption of the blood-brain barrier (BBB) plays a major role in the progression of HAND. However, the underlying mechanisms that regulate the BBB during HIV-1 infection remain poorly defined. In this competitive renewal, we propose to test the hypothesis that BBB permeability is the result of deregulated trans-cellular communication between brain endothelial cells and astrocytes, following enduring effects of HIV-1 proteins on CD40/CD40L signaling. Our project seeks to shift current research and clinical paradigms by defining novel intracellular signaling targets, such as the CD40/CD40L axis and the Sonic hedgehog (Shh) pathway, that play potentially crucial roles in regulating BBB permeability in response to HIV-1 infection, and through functional validation of these targets by using novel anti-Shh drugs as novel therapeutics for HAND. Here we intend to integrate a comprehensive analysis of cellular responses due to HIV-1 with novel methodologies such as intravital multiphoton microscopy in a humanized mouse model. In Aim 1, we will test the hypothesis that CD40/CD40L signaling plays an important role in HIV-1-induced BBB permeability in vivo. Aim 2 will examine whether CD40/CD40L signaling disrupts trans-cellular communication between astrocytes and brain endothelial cells. Finally, in Aim 3 we will investigate whether targeting the Shh pathway renders mice resistant to BBB permeability induced by HIV-1 Tat in a CD40L-dependent manner. The results obtained in these studies are expected to exert a high impact on the neuroAIDS field on three counts: (1) by advancing scientific knowledge and revealing how HIV-1 induces CNS inflammation in infected patients, (2) by promoting novel methodologies and animal models, and (3) by validating novel therapeutic targets for HAND, which are also viable in other neurodegenerative disorders that are secondary to BBB disruption.